Genetic methods have played a major role in efforts to understand the molecular basis for biological phenomena. For example, genetic analysis of the fruit fly, D. melanogastor, provided the entry point for isolation of numerous genes that regulate the formation of the fly body. These genes in turn served as probes for isolation of mammalian homologs that have been the primary tools in molecular studies of vertebrate development.
A variety of genetic and biochemical studies have proved that virtually any biological process (i.e., cell behaviors and the like) can be broken down into components. This reductionist approach to biological inquiry aims to understand the greater part of life's complexity in the relatively simple chemical terms of molecules and molecular interactions. In the middle part of the twentieth century, several scientists, perhaps most notably George Beadle, showed that metabolism can be understood as a series of enzymes that act sequentially to convert precursor compounds into the final metabolic products. This insight gave rise to the notion of genetic or biochemical pathways that control cellular processes. More complicated cellular behaviors such as differentiation have recently been defined in terms of genetic programs and pathways. Even disease processes can be thought of in such terms. For example, cancer is a disease characterized by loss of cellular growth control. An effective strategy to study cancer involves the elucidation of cellular growth regulation pathways. Many genes involved in growth control have been identified and substantial progress has been made in understanding the genetic/biochemical circuitry of these component genes.
Some organisms are especially tractable in genetic studies. These organisms typically are either unicellular, or have short life cycles, small genomes, and a variety of other useful features. Other organisms, such as humans, are less tractable. For tractable experimental organisms, two basic approaches to mutant isolation are available. The first method, termed screening, involves the sometimes painstaking inspection of thousands of individual organisms or clones of cells. Those that have the appropriate mutant phenotype are separated from the others and permitted to grow in isolation. In this manner, homogeneous populations of mutants can be grown and analyzed. The second approach involves growth of organisms under conditions that favor the survival of variant phenotypes over the wild type phenotype. In the case of microorganisms, the selection conditions often involve nutritional requirements or resistance to drugs.
The classical models for genetic studies include E. coli, S. cerevisiae, D. melanogastor, and M. musculus. These organisms share certain features that facilitate genetic studies. First, they can be used to screen and/or select for interesting phenotypic variants (mutants). Second, they can be manipulated in such a way that the underlying genes responsible for specific mutant phenotypes can be localized and isolated by molecular cloning methods. These features permit the analysis of genes in cases where detailed biochemical information about the process under study is unavailable. All that is required at the outset is a tractable experimental organism and a phenotype that can be scored or selected.
In certain organisms such as humans which are of great interest, but in which classical genetic methods of selective breeding cannot be applied, it is still possible to use genetic analysis to identify genes. The techniques are somewhat different and involve retrospective phenotypic and genotypic analysis of kindreds that segregate traits of interest. Such kindreds can be used to determine the approximate location of genes that affect the trait of interest. This approach relies heavily on aspects of heredity that involve sexual reproduction, segregation, and recombination. From rough mapping information, the responsible gene(s) can often be isolated (Mild Y., Swensen J., et al., Science 266: 66-71 (1994)).
Cultured cells from multicellular organisms, as well as single-celled organisms, offer the great advantage that genetic studies can be performed on the simplest unit of life, the cell. In many microorganisms, genetic methods are suitably advanced so that detailed genetic analysis of a wide variety of phenotypic traits is possible. In other organisms such as humans, however, genetic studies in cultured cells are still very difficult. Though cultured somatic cells have provided the route to identification of several important human genes, somatic cells have traits that seriously limit their utility. They are diploid; hence mutants with a recessive phenotype are rarely observed. They reproduce clonally; hence it is not possible generally to map interesting mutations. They are often heterogeneous; hence, each cell in a supposedly identical population of cells may differ slightly in phenotype from another cell for a variety of genetic and epigenetic reasons. They do not lend themselves to a large variety of selection schemes. Genetic methods that can mitigate against these problems in human cells would be particularly valuable.
Genes regulate some of the most medically and commercially important processes in biology. A long list of human diseases are caused by mutations or malfunctions of specific genes. Cancer may be the most familiar example, as it involves the sequential alteration of proto-oncogenes and tumor suppressor genes as tumors progress through stages of malignancy (Fearon E. R. and Vogelstein B., Cell 61: 759-767 (1990)). Methods capable of identifying the underlying genes that regulate important biological processes such as tumor progression would thus be of great value.
For the foregoing reasons, a general method of genetic analysis in cultured cells is needed. The method should be simple, rapid, and permit identification of components of genetic pathways that regulate traits of interest. It should circumvent many of the obstacles that have interfered with genetic analysis in certain cells and organisms. It should not require an understanding of the detailed basis of a particular phenotype, or the mechanisms that underlie specific cellular behaviors. The method should be generally applicable to a great variety of cells, including cells cultured from somatic tissues of multicellular organisms, and it should sidestep certain disadvantages of somatic cell genetics, including the diploid character of most cells, the difficulty of isolating mutant genes once mutations have been induced, and the heterogeneity of many cell populations.